1. Field of Invention
The present invention relates to the controlled modification of peptide sequences with substantially non-antigenic polymeric moieties at one or more selected, predetermined positions.
2. Description of Related Art
U.S. Pat. No 4,904,584 describes modified proteins having naturally occurring amino acids such as lysine replaced with other amino acids to eliminate the random attachment of polyethylene glycol. The homogeneous conjugates are said to eliminate the need to separate the variety of conjugated species of the polypeptide. While the lysine depleted variants in theory would allow site specific polymer attachment, there is no guarantee that the described method actually achieves modification at all selected sites.
The ability to selectively attach moieties to specific sites on proteins and peptides continues. This is particularly the case with small peptides having, for example, fewer than thirty amino acids. These peptide modifications have been particularly troublesome and loss of activity has often resulted.
The covalent attachment of strands of a poly(alkylene glycol) to polypeptides is disclosed in U.S. Pat. No. 4,179,337. This reference discloses that proteins and enzymes modified with poly(ethylene glycols) have reduced immunogenicity and antigenicity and also have longer lifetimes in the bloodstream, compared to the unmodified compounds.
To effect covalent attachment of poly(ethylene glycol) (PEG) to a polypeptide, the hydroxyl end groups of the PEG must first be converted into reactive functional groups. This process is frequently referred to as "activation" and the product is called "activated PEG'. Methoxy poly(ethylene glycol) (mPEG), distally capped with a reactive functional group is often used. One such activated PEG is succinimidyl succinate derivative of PEG (SS-PEG). See also Abuchowski et al., Cancer Biochem. Biophys. 7:175-186 (1984) and commonly assigned U.S. Pat. No. 5,122,614 which discloses poly(ethylene glycol)-N-succinimide carbonate and its preparation.
Chem. Pharm. Bull. 39(12):3373-3375 (1991) discloses hybrids of a fibronectin-related tripeptide (Arg-Gly-Asp) and amino-poly(ethylene glycol) and their inhibitory effect on experimental metastasis in mice. The tripeptide was coupled with amino PEG by activating aspartic acid with dicyciohexylcarbodiimide (DCC)/1-hydroxybenzotriazole (HOBt). This method, however, is only useful for C-terminal modifications. Another limitation of this methodology is that C-terminal activation of peptides promotes racemization of neighboring amino acids. Such modifications would not be observed with glycine, all other amino acids would become racemic with consequent loss of activity.
Journal of Protein Chemistry 10(6):623-627 (199) describes a procedure for synthesis of peptides to monomethoxy poly(ethylene glycol) (mPEG) or polyvinyl alcohol (PVA) using a synthetic resin. This method, however, results in the conjugates having only one poller molecule coupled to the N-terminal of an otherwise unaltered peptide molecule.
While numerous references have reported that PEG conjugates are useful, for example in the medical arts, there often remains the need to control the sites at which the polymer attaches to maintain optimal biological activity. Uncontrolled polymeric conjugation sometimes results in polymeric strands attaching at sites that are critical for biological activity. This disadvantage can be especially acute for polypeptides having relatively short chains, since the possibilities for attachment are limited and the probability of attachment at a critical site correspondingly increases.
Accordingly, a need continues for a process which allows polymeric conjugation to polypeptides, yet avoids the loss of bioactivity.